Bioorganic & Medicinal Chemistry Letters 17 (2007) 2293–2298

Structure-based design of benzylamino-acridine compounds asG-quadruplex DNA telomere targeting agents

Cristina Martins,a Mekala Gunaratnam,a John Stuart,a Vaidahi Makwana,a

Olga Greciano,a Anthony P. Reszka,a Lloyd R. Kellandb and Stephen Neidlea,*

aCRUK Biomolecular Structure Group, The School of Pharmacy, University of London, 29-39 Brunswick Square,

London WC1N 1AX, United KingdombAntisoma Laboratories, St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0QS, United Kingdom

Received 4 December 2006; revised 16 January 2007; accepted 18 January 2007

Available online 25 January 2007

Abstract—The design, synthesis, biophysical and biochemical evaluation is presented of a new series of benzylamino-substitutedacridines as G-quadruplex binding telomerase inhibitors. Replacement of the previously reported anilino substituents by benzyla-mino groups results in enhanced quadruplex interaction, and for one compound, superior telomerase inhibitory activity.� 2007 Published by Elsevier Ltd.

Telomeres are specialized nucleoprotein structures at theends of eukaryotic chromosomes protecting them fromdegradation and recombination, and allow the DNAdamage response to distinguish between double-strandbreaks and natural chromosome ends.1,2 TelomericDNA comprises tandem repeats of simple G-richDNA sequence motifs (TTAGGG in human telomeres),typically several kilobases in length. The extreme 3 0 ter-minus of telomeric DNA (ca. 150–200 nucleotides) issingle-stranded. This single-stranded overhang is associ-ated with the hPOT1 protein,3 which plays a key role inthe regulation of telomere length. Telomeres progres-sively shorten in somatic cells at successive rounds ofreplication. As a consequence of the ‘end-replication’effect, DNA polymerase is unable to fully replicate theblunt ends. When telomeres reach a critically shortlength, cells enter a replicative senescence state and divi-sion no longer occurs.4

All human cancer cells maintain the integrity of telo-mere length.5 Over 80% do so by up-regulation of thereverse transcriptase enzyme telomerase, which catalysesthe synthesis of telomeric DNA repeats,6 thereby stabil-ising telomeres and contributing to cellular immortalisa-tion and oncogenesis.7 The telomerase enzyme complex

0960-894X/$ - see front matter � 2007 Published by Elsevier Ltd.

doi:10.1016/j.bmcl.2007.01.056

Keywords: G-quadruplex; Acridines; Telomerase.* Corresponding author. Tel.: +44 207 753 5969; fax: +44 207 753

5970; e-mail: stephen.neidle@pharmacy.ac.uk

comprises two principal components, a catalytic domain(hTERT) and an RNA template (hTR), and variousapproaches have been devised to target one or other asan anticancer strategy.8 A modified oligonucleotide tar-geting hTR has recently entered Phase I clinical trial.9

Another approach targets the substrate, the 3 0 single-stranded end of telomeric DNA, and uses small mole-cules to induce its folding into quadruplex structures,which are inaccessible to the RNA template componentof the telomerase complex.10 A large number of suchsmall molecules have been reported,11 which bind quad-ruplex DNA and efficiently inhibit telomerase. Most ofthem are based on a polycyclic aromatic core that inter-acts by p–p stacking with the planar G-tetrad motif of aquadruplex,12 and have cationic side chains that interactwith the negatively charged phosphate backbones inG-quadruplexes.

We have previously reported13 on a number of 3,6,9-tri-substituted acridine molecules, one of which, BRACO-19 (compound 1: Table 1), has been studied in detail.It produces cell growth arrest, chromosomal end-to-end fusions,14 and anticancer activity in tumour xeno-grafts,15 all within a short time after exposure, contraryto the paradigm that extended exposure to telomeraseinhibitors is necessary in order to induce anticancer ef-fects. It also, in common with the natural product telo-mestatin,16 competes with hPOT1 for binding to thesingle-strand telomeric DNA overhang, therebyinducing it to fold into a quadruplex structure.17 The

Table 1. Data for FRET-based DTm values (�), telomerase inhibition (lM) and cell growth inhibition (lM) for the trisubstituted acridines

Compound Benzylamine R1 FRET (DTm) TRAP EC50 DU145 MCF7 A549

1 HN N N 28 0.12 2.3 2.53 2.42

2 HNF

FN 29.5 0.03 9.75 2.26 2.11

3HN

OMe

OMe

N 32.5 0.35 10.44 3.72 4.57

4HN N 29.5 1 10 3 1.19

5HN

CF3

F

N 30.5 0.24 2.53 1 2

6HN

CF3

CF3

N 27.5 0.86 2.61 1.07 6.3

7 HNF

FN 26.5 0.88 2.25 0.52 1.2

8HN

OMe

OMe

N 30 0.44 0.47 0.2 0.37

9HN N 31 0.23 0.77 0.46 0.5

10HN N 24 0.39 0.63 0.58 0.57

11 HN N N 21 0.36 2.57 0.54 2

2294 C. Martins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2293–2298

identification of BRACO-19 as a potent G-quadruplexbinding molecule and telomerase inhibitor has led tothe concept of these molecules as Telomere TargetingAgents, whose selective action is due to the uncappingof telomerase from telomere ends, resulting in the inductionof a rapid DNA damage response and selective cell death.

N

HN

NH

NH

OO

N

NMe2

1

NH H

H+

++

N

HN

NH

NH

OO

N NH H

H+

++

2

FF

This report describes an approach to maximizingquadruplex target affinity and biological activity for3,6,9-trisubstituted acridines by rationally altering akey structural feature in these molecules, the 9-substi-tuent, which in BRACO-19 and analogues is an ani-line group. We have used as a starting-point forstructure-based design the crystal structure of thepotassium form of the intramolecular 22-mer humanquadruplex,18 which has an all-parallel topology forthe backbone.19 This structure, with its propellerloops and large G-tetrad surface area (the G-plat-form), has been used to represent the quadruplex foldformed from the single-stranded telomeric DNA over-hang in the crowded environment of the cell nucleus,in accord with observations that the all-parallel formpredominates in polyethylene glycol solution.20 Wecannot exclude the possibility of other topologiesfor the human intramolecular quadruplex, such asthe mixed parallel–antiparallel arrangement reportedin NMR studies,21 although the sequences analysedin these studies have been modified from wild-typetelomeric repeats.

C. Martins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2293–2298 2295

A simulated-annealing and molecular dynamics proto-col22 was used to explore possible ligand binding modesand located a plausible low-energy binding geometry forcompound 1 (BRACO-19) with the quadruplex struc-ture. This geometry for an acridine-quadruplex complexis distinct from that reported previously13 using the anti-parallel NMR-derived quadruplex structure23 and has a6.6 kcal mol�1 lower free energy of binding. In this newarrangement the central acridine core stacks on two gua-nines of the G-platform and both the 3- and 6-sidechains straddle the second loop of the quadruplex. Thecationic pyrrolidino end-groups are held in the bindingpockets formed by the TTA loops of the quadruplex.The 9-substituent N-dimethylanilino group is non-co-planar with the acridine ring due to steric hindranceand is orientated out of the plane of the G-platform(Fig. 1). We envisaged that adding an additional methy-lene linker at the 9-position would enable the benzyl ringto be more coplanar with the G-platform (Fig. 1), there-by improving its affinity to human telomeric quadruplexDNA. The effects of substituents on the benzyl ring suchas fluorine or methoxy on quadruplex affinity wereexplored, to ascertain if they would help to counterthe steric effects of the non-planar methylene bridge. Itwas also reasoned that judicious choice of substituentswould enhance the lipophilicity and hence uptake ofthese polar compounds and improve their pharmacoki-netic features. Fluorine substitution would also improvestability against oxidative metabolism. Moleculardynamics simulations of the G-quadruplex complexesfor 1 and 2 were undertaken using the AMBER pack-age24 and binding energies calculated using the Pois-son-Boltzmann continuum solvent model. This gavethe relative binding free energies of 1 and 2 as�25.7 ± 0.2 and �28.4 ± 0.2 kcal mol�1, respectively.

In order to test the benzylamine hypothesis and generatesome further compounds for subsequent pharmacologi-cal evaluation, a small focussed library of ten 3,6,9-ben-zylamino derivatives has been synthesised as describedin Scheme 1.26 Only the most conservative of changeswere made to the 3,6-side chains, in accord with previ-ous structure–activity studies,13 which have found thatthe pyrrolidino- and piperidino-propionamido substitu-ents are of optimal size and chain length.

Figure 1. Solvent-accessible surface representations25 of the low-energy quad

simulations. Carbon atoms of the ligand are coloured green. The complex wit

ca. 45� to the G-platform. The complex with compound 2 is on the right, w

Variation of substituents on the benzyl ring was restrict-ed to simple electron-donating and withdrawing groups.A detailed synthetic route to these 3,6,9-trisubstitutedacridines has been described previously13 with the excep-tion of the final step, which has been modified for thepresent compounds, enabling the final product to beprecipitated out in the salt form (Scheme 1). All finalcompounds were isolated as the free bases from a sol-vent mixture of DCM/dilute ammonia and were subse-quently used as such for biophysical and biologicalstudies.

All compounds were evaluated for their ability to bindand stabilise G-quadruplex structures by a modifiedfluorescence resonance energy transfer (FRET) as-say27,28 using the 21-mer d[GGG(TTAGGG)3] oligonu-cleotide end-labelled with a fluorescent donor–acceptorpair, and the change in donor/acceptor excitation emis-sion monitored with respect to temperature. The result-ing DTm values provide an indication of the stability of agiven ligand-quadruplex complex. Human telomeraseenzyme inhibition was measured in a modified telome-rase repeat amplification protocol (TRAP) assay,29

and results are expressed as telEC50 values. Compoundswere also evaluated for in vitro cell growth inhibition,expressed as IC50 values, using the sulforhodamine B(SRB) assay in three human tumour cell lines, DU145(prostate), MCF7 (breast carcinoma) and A549 (non-small cell lung carcinoma). Results are presented in Ta-ble 1, which also shows the structures of individualmembers of the series.

In general all of the compounds are effective G-quad-ruplex stabilisers. They produced changes in DTm inthe range of 21.0–32.5� at a ligand concentration of1 lM. Data at other ligand concentrations (availableas Supplementary Data) show the same overall trends.The majority of compounds produced an increase inDTm values when compared to compound 1 (BRAC-O-19), with 3 showing the greatest ability to stabilisehuman G-quadruplex DNA, with a difference inDTm of 4.5� at 1 lM compared to 1. This broadlysupports the hypothesis that the effects of the benzylgroup at the 9-position result in enhanced quadruplexaffinity. Compounds 3 and 8, with –OMe substituents,

ruplex-ligand complexes from simulated annealing/molecular dynamics

h compound 1 is on the left, with the N-dimethylanilino ring orientated

ith the benzyl ring stacked approximately parallel to the G-platform.

NO2

NO2O2N

NO2 NO2

NO2O2N

NO2 O

R1 R1NH

NH

O

NH

O

O

R1 R1N NH

O

NH

O

Cl

NH2NH2

O

NH

NHN

H

O

NHCl

O O

Cl

DMA, NaCO3

R1 R1N NH

O

NH

O

NH

R

82%

KNO3/H2SO4 CrO3/AcOH

94%

HCl/SnCl262%

secondary amine

POCl3

85%

70%

3-CPC, 88%

1) CH3CN, DIPEA, Amine

2) DCM/ammonia

Scheme 1. Synthetic route to the benzylamino-substituted acridines. In the final step, 8.0 g (16.2 mmol) of the 9-chloro was suspended in 250 mL

acetonitrile. 2.6 g (17.8 mmol) DFBA and 2.3 g (17.8 mmol) DIPEA were added and the mixture heated to reflux for 28 h. The solid was collected by

filtration of the hot reaction mixture and washed twice with hot acetonitrile and dried under vacuum. The product was obtained as a yellow solid in

(8.32 g) 87%.

2296 C. Martins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2293–2298

are more effective than 2 and 7, with strongly elec-tron-withdrawing –F substituents, suggesting that theeffects are both steric and electronic. The greater bulkwhen two –CF3 groups are present, in compound 6 inparticular, has a deleterious effect on binding (shownby, for example, compound 5 > 2 > 6), as does thereplacement of the pyrrolidino rings at the 3,6-posi-tions with piperidino ones. An exception to this isthe superior stabilising ability of 9 compared to 6;the reasons for this are not apparent. Even so, com-pounds 7 and 8 are significantly superior quadruplexbinders than the piperidino analogue of BRACO-19,compound 11. Molecular modelling suggests that thepiperidino are slightly too large for optimal fitting intothe quadruplex grooves (Fig. 1); compound 10, whichalso has an extra methyl substituent on this ring, isunsurprisingly a weaker binder and is the least potentcompound in this group. Compounds 4 and 9 are aspotent quadruplex binders as 2 indicating that thepresence of the methyl group on the benzyl ring canbe tolerated, in accord with the structural model. Ta-ble 1 shows that benzylamino substituents in the 9-po-sition bearing electron-withdrawing groups (such as 2and 5) have lower DTm values than the analoguesbearing electron-donating groups. This is to be expect-ed since the benzyl ring lies close to the cationic chan-

nel of the quadruplex, which would thus contribute toits binding.

All of the compounds in this series inhibit telomerasewith telEC50 values <1 lM. There is no simple correla-tion between telEC50 and DTm values, or with short-termcell kill (IC50 values). As previously observed13 effectivequadruplex stabilisation is a necessary element but by it-self is not sufficient for telomerase inhibitory potency. Italso has to be borne in mind that DTm values are notequivalent to association constants, and so high correla-tions with biochemical and biological measures of activ-ity should not be expected. Even so, it is apparent thathigh DTm values are consistently associated with telome-rase potency as estimated by telEC50 values. The mostpotent telomerase inhibitor, compound 2, has given anincreased DTm value compared to 1, of 1.5�, but this issignificantly less than that for 3. The increased short-term cell kill potency observed for the piperidino com-pounds 7–10 compared to their pyrrolidino counterparts2–4 appears to be due to the increased lipophilicity ofthe former group, and is not related to telomerase inhi-bition. The calculated logP values for 2 and 7, for exam-ple, are 1.59 and 2.35, and are a consequence of theadditional two carbon atoms at the ends of the sidechains.

C. Martins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2293–2298 2297

The favourable DTm and telEC50 values for compound 2compared to 1 (BRACO-19), together with its improvedlipophilicity and pharmacokinetic behaviour (to be pub-lished), have led to the selection of 2 as a potential clin-ical candidate molecule. In vivo antitumour data for 2will be reported elsewhere, but off-target toxicity hasbeen more recently observed, and further compoundswith diminished toxicity are now being evaluated.

Acknowledgments

We are grateful to Cancer Research UK and AntisomaLtd for support of these studies.

Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.bmcl.2007.01.056.

References and notes

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26. Compound 2: dH (400 MHz, CDCL3) 1.85 (br s, 8H), 2.50(t, 5.6 Hz, 4H), 2.63 (br s, 8H), 2.80 (t, 5.6 Hz, 4H), 4.81 (s,2H), 7.04 (m, 2H), 7.19 (m, 1H), 7.87–7.89 (m, 6H), 11.50(s, 2H); m/z 602 (MH+); C34H38N6O2F2 requires 601.3097;observed 601.3080. Compound 3: dH (400 MHz, MeOD)1.86 (s (br), 8H), 2.51 (t, 5.6 Hz, 4H), 2.64 (br s, 8H), 2.81(t, 5.6 Hz, 4H), 3.68 (s, 6H), 4.80 (s, 2H), 6.34 (m, 1H),6.46 (m, 2H), 7.07 (m, 2H), 7.82 (m, 2H), 7.91 (d, 9.2 Hz,2H), 11.52 (s, 2H); m/z 625 (MH+); C36H44N6O4 requires625.3497; observed 625.3500. Compound 4: dH (400 MHz,CDCL3) 1.92 (br s, 8H), 2.35 (s, 3H), 2.57 (t, 5.6 Hz,4H),2,69 (br s, 8H), 2.87 (t, 5.6 Hz, 4H), 4.89 (s, 2H), 7.13–7.20 (m, 3H), 7.24–7.28 (m, 1H), 7.74 (m, 2H), 7.88 (br s,2H), 7.99 (d, 9.2 Hz, 2H) 11.50 (s, 2H); m/z 579 (MH+);C35H42N6O2 requires 579.3442; observed 579.3464. Com-pound 5: dH (400 MHz, CDCL3) 1.94 (s (br), 8H), 2.58 (t,5.6 Hz, 4H), 2.72 (br s, 8H), 2.88 (t, 5.6 Hz, 4H), 5.03 (s,2H), 7.43–7.96 (m, 9H), 11.63 (s, 2H); m/z 651 (MH+);C35H38N6O2F4 requires 651.3065; observed 651.3049.Compound 6: dH (400 MHz, CDCL3) 1.94 (s, 8H), 2.58(t, 5.6 Hz, 4H), 2.71 (s, 8H), 2.88 (t, 5.6 Hz, 4H), 5.12 (s,2H), 7.82–7.99 (m, 9H), 11.65 (s, 2H); m/z 701 (MH+);C36H38N6O2F6 requires 701.3033; observed 701.3034.Compound7: dH (400 MHz, CDCL3) 1.50 (br s, 4H),1.66 (br s, 8H), 2.52 (br s, 12H), 2.66 (m, 4H), 2.93 (s, 6H),4.93 (s, 2H), 6.84 (d, J = 8.4 Hz 1H), 7.11 (m, 2H), 7.27(m, 1H), 7.85 (br s, 2H), 7.88–7.98 (m, 3H), 8.32 (m, 1H),11.66 (s, 2H); m/z 629 (MH+); C36H42N6O2F2 requires629.3410; observed 629.3429. Compound 8: dH (400 MHz,CDCL3) 1.46 (s (br), 4H), 1.65 (br s, 8H), 2.53 (m, 12H),2.66 (m, 4H), 3.73 (s, 6H), 4.88 (s, 2H), 5.69 (br s, 1H),6.40 (br s, 1H), 6.55 (br s, 2H), 7.82–8.06 (m, 6H) 11.65 (s,2H); m/z 654 (MH+); C38H48N6O4 requires 653.3810;

2298 C. Martins et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2293–2298

observed 653.3821. Compound 9: dH (400 MHz, CDCL3)1.50 (s (br), 4H), 1.68 (br s, 8H), 2.35 (s, 3H), 2.52 (m,12H), 2.66 (m, 4H), 4.90 (s, 2H), 5.48 (br s, 1H), 7.18 (m,4H), 7.83 (br s, 2H), 7.90 (br s, 2H), 7.02 (d, 8.2 Hz, 2H)11.64 (s, 2H); m/z 608(MH+); C37H46N6O2 requires607.3755; observed 607.3786. Compound 10: dH

(400 MHz, CDCL3) 1.47 (s (br), 4H), 1.63 (br s, 8H),2.43 (br s, 12H), 2.58 (m, 4H), 6.67–6.85 (m, 4H), 7.15 (brs, 2H), 7.82 (m, 2H); m/z 622 (MH+); C37H47N7O2 requires622.3863; observed 622.3859. Compound 11: dH

(400 MHz, CDCL3) 1.52 (br s, 4H), 1.68 (br s, 8H), 2.54(br s, 12H), 2.66 (m, 4H), 2.93 (s, 6H), 6.72 (d, J = 8.8 Hz2H), 6.92 (d, J = 8.4 Hz, 2H), 7.23 (m, 2H), 7.94 (m, 4H),11.63 (s, 2H); m/z 623 (MH+); C37H47N7O2 requires622.3864; observed 622.3859.

27. Schultes, C. M.; Guyen, B.; Cuesta, J.; Neidle, S. Bioorg.Med. Chem. Lett. 2004, 14, 4347.

28. FRET experiments: HPLC purified and desalted oligonu-cleotide F21T was supplied by Eurogentec. It wasannealed at a concentration of 400 nM in buffer (50 mMKCl, 10 mM cacodylic acid, adjusted to pH 7.4 by KOHaddition) by heating to 92� for 2 min and allowing to coolto room temperature over 4 h after which it was furtherdiluted to 317 nM with buffer. Stock solutions of ligandswere prepared in potassium chloride-cacodylate buffer. 96-well plates (Bio-Rad MLL9651) were prepared with aGilson Cyberlab C-200 Robotic Workstation, each con-taining 100 lL aliquots per well with an oligonucleotideconcentration of 200 nM and a concentration seriesbetween 0.10 and 10.00 lM for each ligand. The wellswere read in a DNA Engine Opticon 1 real-time PCRcycler (MJ Research). Fluorescence was measured at 0.5�intervals whilst increasing the temperature from 30� to100� at a rate of 1�/min. Excitation was at 450–495 nm anddetection at 515–545 nm. Data analysis was performedusing Origin Pro 7.0 (OriginLab Corporation). Experi-ments were performed in triplicate and esds are ±0.5�.

29. TRAP assay: the telomerase repeat amplification protocol(TRAP) assay was used to assess the activity of telomerase

in the presence of the compounds. It was conducted usinga modified version of standard published TRAP proto-cols.24 The telomerase enzyme source was a cell extractfrom exponentially growing A2780 human ovarian carci-noma cells. The TRAP assay was carried out in two steps.First, for the elongation of the primer by telomerase, aTRAP reaction mixture (40 lL) was prepared, containingthe TS forward primer (0.1 lg; 50-AATCCGTCGAGCA-GAGTT-3 0; Invitrogen, Paisley, UK), TRAP buffer(20 mM Tris–HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl21 mM EGTA and 0.05% v/v Tween 20), BSA (0.05 lg) anddNTPs (125 lM). Protein (1 lg) was then incubated withthe TRAP reaction mixture with or without drug for10 min at 30�. Subsequently, the telomerase was inacti-vated by heating to 92� for 4 min and cooling to 10�.During cooling, 10 lL of a PCR mixture containing ACXprimer (0.1 lg; 50-GTG[CCCTTA]3CCCTAA-3 0; Invitro-gen, Paisley, UK) and Taq polymerase (RedHot, AAB-gene, Surrey, UK) was added to each tube, thus initiatingthe second step of amplification of the telomerase prod-ucts. The polymerase chain reaction was carried out in 33cycles of 92� for 30 s, 55� for 30 s and 72� for 45 s. Finally,the amplified PCR products were run out on a 10% w/vPAGE gel and visualized by staining with SYBR Green I(Sigma). The TRAP products were quantified by fluores-cence and the telEC50 values were calculated using Gene-Tools software (Syngene, Cambridge, UK) andOriginPro 7.0 (Originlab Corp., Northampton, MA).Experiments were performed in triplicate and esds areestimated to be ±0.02 lM. All compounds were exam-ined for their ability to inhibit the activity of TAQpolymerase, and no inhibition was observed at or nearthe concentrations required for telomerase inhibition.Results from the TRAP assay performed in differentlaboratories cannot readily be compared since differingexperimental protocols are frequently used, and theresults presented here should be considered as relativerather than as absolute indicators of telomeraseinhibition.